Biomarker of renal dysfunction

ABSTRACT

The present invention relates to a method for a method for predicting the development of renal dysfunction in a subject following physical trauma, hypotension, sepsis and/or septic shock syndrome, wherein the method comprises the steps of: —a. determining the level of an anti-inflammatory cytokine present in a sample taken from the subject after physical trauma, after a hypotensive event, after sepsis, and/or after septic shock syndrome; b. predicting the development in the subject of renal dysfunction on the basis of the level of an anti-inflammatory cytokine determined in step a).

The present invention relates to a method for predicting the developmentof renal dysfunction, and to a kit for use in making such a prediction.

Apart from the direct adverse impact of physical trauma on the body(e.g. from lesions caused by surgery or car crashes, or from surgicalprocedures such as blood bypass through a heart-lung machine), subjectssuffering physical trauma often develop acute renal dysfunction. Othercauses of a similar acute renal dysfunction include prolongedhypotensive states (e.g. associated with mucosal gut ischaemia andendotoxin translocation from gut to circulation), sepsis and septicshock syndromes.

In a population of patients who have normal renal function, diagnosis ofpost-trauma, hypotensive-induced, sepsis-induced, and/or septic shocksyndrome-induced renal dysfunction can be made on the basis of a fall inGlomerular Filtration Rate (GFR) as compared with the normal or baselineGFR; as measured, for example by the MDRD test (explained in more detailbelow). The limitation of a diagnosis or prognosis based on suchmeasurements is that it usually takes several days for MDRD estimatedGFR values to fall. When the GFR fall is detected, it is often too lateto institute therapeutic measures to obviate further deterioration inrenal function. The challenge facing for example the peri-operativephysician or intensive care physician is to identify a biological markerof renal dysfunction 48 hours before such dysfunction materialises (i.e.while there is still a possibility of preventative measures being takenin the intensive care unit; such as running such patients onsupra-normal blood pressures, an intervention which in its own right isnot without risk in the postoperative context, unless justified by thepresence of an even greater risk of impending renal failure such ascould be identified by the test according to the present invention).

It is well documented that trauma to the body induces an acute (oftentransient) plasma pro-inflammatory response. It has been shown that themagnitude of this acute plasma pro-inflammatory response correlates withpost-trauma renal dysfunction (Gormley et al., Anesthesiology, 2000,93). Other causes of a similar acute reno-toxic pro-inflammatoryresponse include prolonged hypotensive states (associated with mucosalgut ischaemia and endotoxin translocation from gut to circulation), orsepsis and septic shock syndromes, all of which are usually eitherpreceded by or associated with a systemic inflammatory response (SIRS)characterised by acute increases in pro-inflammatory mediators in theblood. A mechanism for how such acute pro-inflammatory responses whenfiltered from the blood damage renal tubules was suggested by Chatterjeewho demonstrated in vitro that TNF-[alpha] applied to proximal tubulesin cell culture leads to cell damage (Markewitz, J Clin Invest 1993; 91;Chatterjee et al., Exp Nephrol 1999; 7:438-8). Following challenge withthe combination of inflammatory cytokines IL-1 beta, TNF-alpha, andIFN-gamma, in vitro proximal tubular cells exhibit a time-dependentincrease in inducible NO synthase induction and corresponding NOproduction to cytotoxic concentration, an effect which was inhibited byL-NMMA (Chatterjee et al., Exp Nephrol 1999; 7:438-8). Stimulation ofrat kidney epithelial cells with TNF-alpha and IFN-gamma dramaticallyincreased the level of inducible nitric oxide synthase mRNA (Markewitz,J Clin Invest 1993; 91).

Numerous publications show that levels of urinary anti-inflammatorycytokines show dramatic increases after trauma, and in fact mirror theaforementioned increase in level of pro-inflammatory responses. See, forexample: —Gormley et al. Cytokine, January 2002, 21 17(2). Further tothis, it has been demonstrated that the magnitude of the post-traumaincreases in anti-inflammatory cytokines correlate with the magnitude ofrenal injury and dysfunction. See, for example: —Litalien et al.,Pediatr. Nephrol., November 1999, 13(9); Gretchen et al.,Anesthesiology, November 2000, Gormley et al., Anesthesiology, 2000, 93,and; European Patent Publication No. 1 962 092 A (Renovar, Inc.). It isclear from the presentation of these results in the aforementionedpublications that a relatively large post-trauma anti-inflammatorycytokine level is proposed as a directly proportional marker for renaldysfunction.

Alternative studies have suggested instead that renal dysfunction can bedetermined by measuring the change in the level of anti-inflammatorycytokines from before a physical trauma to after a physical trauma. Thisanalysis obviously requires the sampling and evaluation ofanti-inflammatory cytokine level both before and after a physicallytraumatic event.

The inventors have been surprised to find that when they analysed theanti-inflammatory cytokine responses of a large group of post-traumasubjects that a population of those subjects presented with only arelatively low anti-inflammatory cytokine level following physicaltrauma, as opposed to the expected large anti-inflammatory cytokinelevel previously reported and as experienced by the majority ofsubjects. On further analysis of this subgroup, it was surprising tofind that its members were at elevated risk of developing renaldysfunction when compared to those that showed the expected largeanti-inflammatory cytokine level following physical trauma. Suchfindings are contrary to the previously accepted conclusion that largepost-trauma urinary anti-inflammatory cytokine levels correlate with themagnitude of renal injury and dysfunction.

The inventor's surprising discovery also demonstrates that one canpredict renal dysfunction by sampling and analysis of anti-inflammatorycytokines at only a single point, i.e. after the physical trauma. Suchanalysis is therefore simpler to carry out than studies that indicatethat the measurement of both pre- and post-trauma change ofanti-inflammatory cytokine levels is required. Measuring onlypost-trauma anti-inflammatory cytokine levels is particularlyadvantageous when trying to predict the development of renal dysfunctionfollowing unexpected events such as physical trauma induced by a carcrash; i.e. when measuring pre-trauma anti-inflammatory cytokine levelsis impossible.

The inventors have, following extensive experimentation, providedevidence in support of the concept that by monitoring for a low level ofanti-inflammatory cytokine post-trauma or hypotensive subject, or in asubject suffering from sepsis or septic shock syndrome, one can arriveat a prognostic method for renal dysfunction.

Accordingly, in a first aspect of the present invention, there isprovided a method for predicting the development of renal dysfunction ina subject following physical trauma, hypotension, sepsis and/or septicshock syndrome, wherein the method comprises the steps of: —

-   -   a. determining the level of an anti-inflammatory cytokine        present in a sample taken from the subject after physical        trauma, after a hypertensive event, after sepsis, and/or after        septic shock syndrome;    -   b. predicting the development in the subject of renal        dysfunction on the basis of the level of an anti-inflammatory        cytokine determined in step a).

The method of the present invention predicts the likelihood of thesubject developing post-event (i.e. post-physical trauma,post-hypotensive event, or after the development of sepsis or septicshock syndrome) renal dysfunction. The method is therefore a prognosticmethod. Subjects may be determined to have a greater than normal chanceof developing renal dysfunction when they present with a level ofanti-inflammatory cytokines as determined by step a) that is lower thanthe normal level of anti-inflammatory cytokine. Subjects may bedetermined to have a lower than normal chance of developing renaldysfunction when they present with a level of anti-inflammatory cytokineas determined by step a) that is higher than the normal level ofanti-inflammatory cytokine.

A normal level of anti-inflammatory cytokine is the post-event levelpresented by a control group of individuals that do not have renaldysfunction on the fifth day following physical trauma, following orduring a hypotensive event, following or during sepsis, and/or followingor during septic shock syndrome. All of the control individuals willhave sustained some degree of sub-clinical renal injury because of theaforementioned events, but will not have had clinically significantrenal injury. Determining whether or not an individual has renaldysfunction is a clinical question well within the abilities of theskilled person. However, in the interests of clarity, renal dysfunctionis characterised by a reduction in the capacity to excrete metabolicproducts which accumulate systemically and are detectableclinicopathologically by renal function tests (in progressed states,renal dysfunction may be acute kidney failure, uremia or chronic renaldamage). For example, the MDRD method of determining renal function isdescribed below. The control group may experience the same or similarevent (i.e. the same or similar trauma, hypotensive event, sepsis,and/or septic shock syndrome) to the subject being diagnosed. Thecontrol group may comprise 30 individuals or more. The control group maycomprise 300 individuals or more.

The normal level may be predetermined (ie normal post-event level). Notwishing to be restricted further, but in the interests of clarity, anormal level of IL1ra may be 22700 pgml⁻¹ or above. A normal level ofTNFsr2 may be 20000 pgml⁻¹, or above. A normal level of TNFsr1 may be20000 pgml⁻¹, or above.

Subjects that present with an anti-inflammatory cytokine leveldetermined in step a) that is progressively less than 100% that of thenormal level for the anti-inflammatory cytokine are at progressivelygreater than normal risk of developing renal dysfunction. Thus, when theanti-inflammatory level determined in step a) is 95% or less, 90% orless, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less,30% or less, 20% or less, or 10% or less that of the normal level thesubject is at greater than normal risk of developing renal dysfunction.Such subjects may be at greater than normal risk of having renaldysfunction 5-days following physical trauma, following or during ahypotensive event, following or during sepsis, and/or following orduring septic shock syndrome.

If the level of IL1ra determined in step a) is less than 12000 pgml⁻¹,it would be predicted that the subject will develop renal dysfunction.If the level of TNFsr2 determined in step a) is less than 11500 pgml⁻¹,it would be predicted that the subject will develop renal dysfunction.

Renal dysfunction induced by physical trauma, hypotension, sepsis and/orseptic shock syndrome may be further characterised by pro-inflammatoryresponses that are induced following these events. Such inflammatoryresponses commonly follow the pattern of a classical systemicinflammatory response (SIRS). Thus, the renal dysfunction to beprognosed according to the present invention may be those that areinduced by an acute (possibly transient) urinary pro-inflammatoryresponse (for example, elevated urinary levels of IL-18 and/or ofneturophil gelatinase-associated lipocalin (NGAL)).

The renal dysfunction to be prognosed may be early renal dysfunction,late renal dysfunction, or general renal dysfunction. Early renaldysfunction occurs within two days of the event that induces the renaldysfunction. Late renal dysfunction occurs 5 days or later after such anevent Renal dysfunction may be defined as a 25% or more decrease fromnormal glomerular filtration rate. Normal glomerular filtration rate isthe pre-even rate. Glomerular filtration rate may be established inaccordance with the MDRD study group formula. Such acute forms of renaldysfunction can be distinguished from autoimmune mediated chronic renaldysfunction, a condition that is clinically apparent over a prolongedperiod of time in parallel with the coexisting autoimmune condition(i.e. there is no requirement for a biological marker to predict thedevelopment of renal dysfunction occurring a few days later because therenal dysfunction is already well established).

The sample taken from the subject may be any sample capable of beinganalysed for the level of anti-inflammatory cytokine therein. Forexample, the sample may be a urine sample, a blood sample or a plasmasample. A urine sample is particularly preferred.

The sample analysed in step a) may be obtained from the subject within24 hours of the event (i.e. physical trauma, hypotensive event, onset ofsepsis and/or onset of septic shock syndrome). The sample will optimallybe obtained within 12 to 24 hours of the event

Step a) may be preceded by a step of obtaining a sample from thesubject.

The predicting of the development in the subject of renal dysfunction inthe present invention is based on the determination of the level of ananti-inflammatory cytokine present in a sample after the recited events.Consequently, it would be clear that the level of an anti-inflammatorycytokine present in a sample after the recited events may be the onlycytokine level required to be determined in order to make the predictionaccording to the present invention. It is also therefore clear that themethods of the present invention do not require the level of ananti-inflammatory cytokine present in a sample prior to the recitedevents to be determined in order to make the prediction according to thepresent invention.

A physical trauma is the impact on the body from external forces appliedto the body, for example: —lesions caused by surgery or by blows or cutsto the body (such as might be experienced during a car crash), or; theimpact on the blood as it interacts with the foreign surface of aheart-lung bypass machine. Renal dysfunction induced by physical traumamay be postoperative renal dysfunction. The post-operative renaldysfunction may be following cardiac, cardiovascular or cardiopulmonarysurgery.

Whether or not an individual has hypotension is a clinical question andtherefore well within the skill of an ordinary person in the art. Forthe avoidance of doubt however hypotension in adults may be defined as asystolic blood pressure <80 mmHg, or a mean arterial pressure (MAP)<50mmHg. The hypotension may be prolonged, for example for over 2 hours.

Whether or not an individual has sepsis is a clinical question andtherefore well within the skill of an ordinary person in the art. Forthe avoidance of doubt however, sepsis may be considered present ifinfection is highly suspected or proven and two or more of the followingsystemic inflammatory response syndrome (SIRS) criteria are met:

-   -   1. Heart rate >90 beats per minute (tachycardia);    -   2. Body temperature <36° C. (97° F.) or >38° C. (100° F.)        (hypothermia or fever);    -   3. Respiratory rate >20 breaths per minute or, on blood gas, a        P_(a)CO₂ less than 32 mm Hg (4.3 kPa) (tachypnea or hypocapnia        due to hyperventilation); and    -   4. White blood cell count <4,000 cells/mm³ or >12,000 cells/mm³        (<4×10⁹ or >12×10⁹ cells/L), or greater than 10% band forms.

Whether or not an individual has septic shock is a clinical question andtherefore well within the abilities of an ordinary person skilled in theart. For the avoidance of doubt however, septic shock may be defined bythe presence of the following two criteria:

-   -   1. Evidence of infection, through a positive blood culture; and    -   2. Refractory hypotension—hypotension despite adequate fluid        resuscitation and cardiac output. In adults it is defined as a        systolic blood pressure <90 mmHg, or a MAP <60 mmHg, before        institution of required resuscitative inotropic support, or a        reduction of 40 mmHg in the systolic blood pressure from        baseline. In children it is BP<2 SD of the normal blood        pressure.

Whether or not an individual has SIRS is a clinical question andtherefore well within the abilities of an ordinary person skilled in theart. For the avoidance of doubt however SIRS may be diagnosed when twoor more of the following are present:

-   -   1. Heart rate >90 beats per minute    -   2. Body temperature <36 or >38° C.    -   3. Tachypnea (high respiratory rate) >20 breaths per minute or,        on blood gas, a P_(a)CO₂<4.3 kPa (32 mm Hg)    -   4. White blood cell count <4000 cells/mm³ or >12000 cells/mm³        (<4×10⁹ or >12×10⁹ cells/L), or the presence of greater than 10%        immature neutrophils.

The anti-inflammatory cytokine may be any cytokine capable ofsuppressing inflammatory responses in the body and that can be detectedin a sample (e.g. the urine). The anti-inflammatory cytokine may be onelocally released in renal tissue. The anti-inflammatory cytokine may beTNFsr1, TNFsr2 or IL-1 ra.

The method may involve analysis of the levels of more than one type ofanti-inflammatory cytokine. For example, the methods according to thepresent invention may further comprise the steps of: —

-   -   a′) determining the level of one or more additional        anti-inflammatory cytokine present in a sample taken from the        subject after physical trauma, after a hypotensive event, after        sepsis, and/or after septic shock syndrome;    -   b′) predicting the development in the subject of developing        renal dysfunction on the basis of the level of an        anti-inflammatory cytokine determined in step a) and a″).

The risk of the subject developing renal dysfunction is greater whenboth (i) the level of anti-inflammatory cytokine is determined by step(a) is lower than the normal level, and (ii) the level ofanti-inflammatory cytokine as determined by step (a′) is lower than thenormal level.

Any combination of the aforementioned anti-inflammatory cytokines may beused in such a method. For example the anti-inflammatory cytokinedetermined in step a) may be IL-1 ra and the one or moreanti-inflammatory cytokine determined in step a′) may be TNFsr2.

Pro-inflammatory markers of impending renal dysfunction have beenidentified; pro-inflammatory mediators NGAL and IL-18 (Mishra et al.,Lancet, 2005; 365:1231-38). Elevations of these substances in the urineof children have been associated with heightened risk of developingrenal dysfunction several days later. Based on the hypothesis presentedby the present inventors, however, establishing that an individual hasan elevated pro-inflammatory response alone may not be sufficient toestablish the risk of developing renal dysfunction.

The method may involve the post-event analysis of the levels of one (ormore than one type of) anti-inflammatory cytokine and one or morepro-inflammatory mediator.

The pro-inflammatory mediators may be any mediator capable of inducingan inflammatory response in the body, that is implicated in thedevelopment of renal dysfunction and that can be detected in the urine.The pro-inflammatory mediators may be IL-18 and/or NGAL.

In a further aspect of the present invention, there is provided a kitfor use in the methods of the preceding claims, wherein the kitcomprises: —

(a) one or more reagents for the detection of the amount of one or moreanti-inflammatory cytokine;

(b) instructions for using the one or more reagents for detecting theone or more anti-inflammatory cytokines.

The kit may further comprise one or more reagents for the detection ofone or more pro-inflammatory mediator, and instructions for using theone or more reagents for detecting the one or more pro-inflammatorymediator.

The kit may further comprise instructions for using the detecting of theone or more anti-inflammatory cytokines in order to arrive at aprognosis for renal dysfunction. The instructions may be in accordancewith the steps for prognosis of renal dysfunction provided in the firstaspect of the present invention. The kit may further compriseinstructions for using the detecting of the one or more pro-inflammatorymediator in order to arrive at a prognosis for renal dysfunction. As theinstructions and reagents are to be capable of being used in order topractice the methods described above in accordance with the first aspectof the present invention, all features of the first aspect of thepresent invention may be included in the second aspect of the presentinvention, where the context permits. Consequently, for example, theanti-inflammatory cytokines (or combinations thereof) and thepro-inflammatory mediators mentioned in the first aspect of the presentinvention may be those recited above in the second aspect of the presentinvention.

In a third aspect of the present invention there is provided a method oftreating renal dysfunction induced by physical trauma, hypotension,sepsis and/or septic shock syndrome, wherein the method includes thesteps of: (i) prognosing renal dysfunction according to any of themethods of the first aspect of the present invention; and (ii) when thesubject is identified to be at increased risk of developing renaldysfunction, applying therapeutic measures to treat or obviate theimpending renal dysfunction. The advantage of such a method over currenttherapeutic interventions is that therapy may be administered at a stagewhen full renal failure may be prevented. The therapeutic measuresapplied in step (ii) may be: maintaining a supra-normal blood pressure;ensuring adequate tissue oxygen delivery; administration of steroids;renal replacement therapy; dialysis; or any combination thereof. Afurther advantage of this invention would be to allow intensive caremanagers to identify early in the intensive care stay of the patientthose individuals who are likely to spend longer in intensive care thanwould otherwise be anticipated providing earlier planning for staffdeployment.

A number of lists are provided above in which optional features for eachof the aspects of the present invention are provided. Each member of thelist is contemplated individually as a potential feature of the presentinvention.

The present invention will now be described, by way of example, withreference to the accompanying figures, in which: —

FIG. 1 shows receiver/operator characteristics (ROC) curve forpost-operative urinary TNFsr2 showing sensitivity and specificity todistinguish patients with and without renal dysfunction as defined byMDRD eGFR drop at day 5 post-surgery of more than 25% from thepre-surgery value.

FIG. 2 shows receiver/operator characteristics (ROC) curve forpost-operative urinary IL1ra showing sensitivity and specificity todistinguish patients with and without renal dysfunction as defined byMDRD eGFR drop at day 5 post-surgery of more than 25% from thepre-surgery value.

FIG. 3 shows a table demonstrating the number of patients on each day ofthe study discussed below who had normal renal function (NF), or renaldysfunction (RD) as defined by greater than 25% MDRD drop from base lineon that day, or for whom renal function data was not know (NK).

EXPERIMENTAL METHODS

Four hundred and eight consecutive patients undergoing elective cardiacsurgery were studied. The patients were recruited within the CardiacSurgical Unit of the Royal Victoria Hospital Belfast in Northern Ireland(n=304) and Papworth Hospital NHS Foundation Trust in Cambridge, England(n=104). Local ethical committee approvals were received and writteninformed patient consent was obtained. Exclusion criteria includedpre-operative dialysis dependent renal failure, significant renaldisease (eGFR <40 ml min⁻¹) or diabetes mellitus. Patients onpre-operative angiotensin conversion enzyme (ACE) inhibitor therapy werenot excluded from this study. The anaesthetic technique in both centerswas based on the use of propofol and fentanyl. Isoflurane was used inmost patients either as an adjunct anaesthetic agent or to control bloodpressure. Pancuronium was used to provide muscle relaxation.Post-operative analgesia was with morphine infusion.

In the 408 elective cardiac surgery patients 24 hour post-operativeurinary IL1ra and TNFsr2 was measured. The post-operative urinaryresponse was compared for each cytokine in patients grouped according topresence or absence of renal dysfunction defined as a drop from baselineestimated glomerular filtration rate (eGFR) of greater than 25% (ascalculated by the method of modification of diet in renal disease(MDRD)) occurring within (1) the first 24 and (2) 48 postoperative hours(early renal dysfunction), (3) on the fifth postoperative day (laterenal dysfunction) or (4) at any time throughout the 5 daypost-operative period (early and late combined)).

Cytokines Analysis (ELISA)

Cytokines are measured by R&D systems Quantikine solid phase ELISAtechnique. This system consists of a conjugate, standard, assay diluent,calibrator diluent, wash buffer concentrate, colour reagent A, colourreagent B, and a stop solution. Reagents should be at the roomtemperature before beginning the assay. The microplate-consists of 96wells. This microplate is coated with capture antibody. To each wellassay diluent is added. Standards in duplicate and the samples are addedto the plate and incubated for 2 h at room temperature. Any analytepresent in the sample is bound by the capture antibody (immobilizedantibody). After the incubation, the plate is aspirated and washed fourtimes with the supplied wash buffer to washout any unbound materials.After washing, horseradish protease (HRP) labelled detection antibody(conjugate) is added to the plate and further incubated at roomtemperature. Once again, after the incubation the plate is aspirated andwashed 4 times. Any unbound detection antibody is washed away. In thenext step prepared substrate solution tetramethylbenzidine (TMB) isadded to the wells and a blue colour develops in proportion to theamount of analyte present in the sample. After 20 minutes incubation thecolour develops (blue) proportional to the cytokine concentration. Foranalysis colour development is stopped turning the colour in the wellsto yellow. The absorbance of the colour at 450 nm is measured which isread in the microplate reader.

Urinary samples for measurement of urinary IL-1ra and TNFsr2 wereobtained at 24 hours after cessation of cardiopulmonary bypass orrevascularisation.

Measurement of Renal Dysfunction

In 1989 Kopple et al as part of the Modification of Diet in RenalDisease study group published their findings investigating the impact ofnutritional status on chronic renal insufficiency in 95 patients. Theeffects on progression of renal disease of a control diet of only milddietary protein restriction were compared with 3 study diets of varyingdegrees of protein restriction and reduced phosphorus intake. Theauthors found that malnutrition and lower energy intake characterisedpatients with the lower GFR levels. There were some gender differenceswith men demonstrating a correlation between GFR and arm muscle area andpercentage standard body weight especially at the onset of theexperimental diets. In women, GFR correlated with dietary energy intake[Kopple et al., Kidney Int Suppl 1989; 27]. A logical development ofthis study from the MDRD group was the idea that analysis of patients'age weight, gender and ethnicity together with serum creatinine wouldallow GFR to be estimated. This assumption recognised that serumcreatinine concentration alone does not adequately reflect renalfunction but should be considered along with the factors identified asinfluencing renal function in Kopple's study.

To develop the prediction equation 1628 patients were enrolled in thebaseline period, of which 1070 were randomly selected as the trainingsample whereas the remaining 558 patients constituted the validationsample. The authors then used stepwise regression to the training sampleto develop the equation which was then tested and compared with theCockcroft and Gault formula and creatinine clearance measurements in thevalidation sample.

It was found that several measured variables were associated with alower GFR. These included higher serum creatinine, higher serum urea andlower serum albumin levels concentrations. Independent variablesassociated with lower GFR included older age group, female gender andnon-black ethnicity (P<0.001 for all factors).

The multiple regression models explained 90.3% of the variance in thelogarithm of GFR in the validation sample. Measured creatinine clearanceoverestimated GFR by 19%, and creatinine clearance predicted by theCockcroft-Gault formula overestimated GFR by 16%. After adjustment forthis overestimation, the percentage of variance of the logarithm of GFRpredicted by measured creatinine clearance or the Cockcroft-Gaultformula was 86.6% and 84.2%, respectively.

MDRD study group estimated GFR is calculated from the following formula:X=32788×creatinine-1.154×age-0.203×constant where the constant is 1 forwhite males, 0.724 for females, and 1.21 for African Americans.

MDRD estimated GFR in the present study was calculated from the aboveformula. According to the above formula MDRD GFR was calculated at preopday 0 and at post operative days 1, 2 and 5. For each separate post opday as well as all post op days together patients can be divided intonormal and abnormal renal function groups where “normal” and “abnormal”were defined by those who sustained falls in MDRD GFR of less than orgreater than 25% of baseline respectively.

In summary then for post surgery cytokine values were compared betweenthe normal and abnormal groups where normality and abnormality weredefined according to the 4 definitions mentioned below.

-   Definition 1: ‘Abnormality’ is defined as having day 1 MDRD >25%    drop from baseline. (Early renal dysfunction).-   Definition 2. ‘Abnormality’ is defined as having day 2 MDRD >25%    drop from baseline. (Early renal dysfunction).-   Definition 3. ‘Abnormality’ is defined as having day 5 MDRD >25%    drop from baseline. (Late renal dysfunction).-   Definition 4. ‘Abnormality’ is defined as having at least one MDRD    value >25% drop from baseline during days 1, 2, and 5. (General    renal dysfunction).    Statistical Methods

Quantitative variables were summarized as mean and standard deviationexcept for those with heavily skewed distributions for which median andinterquartile range were used. Comparisons between groups were obtainedusing the independent samples z-test or Mann-Whitney U test forquantitative variables, and the chi-squared test or Fisher's exact testfor categorical variables. The ability of cytokine measurements todistinguish patients with and without renal dysfunction in thepostoperative period was examined using the receiver-operatorcharacteristic (ROC) curve. The area under this curve provides a measureof discriminatory ability; a value of 1 indicates perfect discriminationwhile a value of 0.5 indicates no more discriminatory ability than wouldbe expected by chance.

Results

1. Numbers of Patients in Renal Dysfunction and their Characteristics

The numbers of patients who developed renal dysfunction on postoperativedays 1, 2 and 5 are illustrated in FIG. 3. Table 1 shows thecharacteristics of patients categorized according to presence or absenceof renal dysfunction defined by >25% drop from baseline eGFR at any timeduring the 5 day postoperative period. Renal dysfunction was more likelyin patients who had increased weight, male gender or who had not beengiven an intra-operative dopamine infusion (Table 1). A requirement forpostoperative resternotomy for bleeding/cardiac tapenade orpost-operative adrenaline infusion was linked with renal dysfunction.Development of renal dysfunction significantly prolonged length of stayin the intensive care/high dependency unit (Table 1).

TABLE 1 The table shows pre- and peri-operative characteristics ofpatients with and without renal dysfunction as defined by >25% drop frombaseline eGFR at any time during the postoperative period (mean +/− SDor n (%)). Patients with MDRD Patients with MDRD eGFR drop ≤25% at alleGFR drop >25% at any P times (N = 349) time (N = 49) value BaselineMDRD eGFR 73.8 +/− 15.3 75.9 +/− 24.5 0.57 (ml/min/1.73 m²) Age (years)62.6 +/− 10.5 65.6 +/− 10.3 0.06 Weight (kg) 77.1 +/− 13.6 82.8 +/− 15.70.02* Height (m) 1.69 +/− 0.10 1.71 +/− 0.08 0.06 BMI (kg/m²) 27.0 +/−4.0  28.2 +/− 4.5  0.08 Male Gender 237/349 (68%) 42/49 (86%) 0.01*Operation time (min) 252 +/− 51  259 +/− 91  0.59 CPB time # (min) 95+/− 34 99 +/− 41 0.52 Cross clamp time †(min) 62 +/− 28 62 +/− 25 0.81Beta blocker 226/349 (65%) 33/49 (67%) 0.72 Calcium Antagonist 83/348(24%) 14/49 (29%) 0.47 Nitrate 162/349 (46%) 20/49 (41%) 0.46 K channelblocker 119/349 (34%) 13/49 (27%) 0.29 ACEI 152/349 (72%) 24/49 (49%)0.47 ATII receptor blocker 13/349 (4%) 4/49 (8%) 0.14 Intra-operative223/349 (64%) 30/49 (61%) 0.72 phenylephrine Any intra-operative 159/348(46%) 18/49 (37%) 0.24 intotrope Intra-operative dopamine 118/348 (34%)7/49 (14%) 0.006* Intra-operative 36/347 (10%) 6/49 (12%) 0.69noradrenaline Intra-operative adrenaline 17/349 (5%) 3/49 (6%) 0.72Intra-operative milrinone 25/349 (7%) 3/49 (6%) 1.00 Intra-operativeIABP 3/348 (1%) 0/49 (0%) 1.00 Any post-operative 156/349 (45%) 20/49(41%) 0.61 inotrope Post-operative dopamine 145/348 (42%) 16/49 (33%)0.23 Post-operative 26/349 (7%) 7/49 (14%) 0.16 noradrenalinePost-operative adrenaline 17/349 (5%) 6/49 (12%) 0.05* Post-operativemilrinone 28/349 (8%) 2/49 (4%) 0.56 Intra-operative IABP 1/349 (0%)0/49 (0%) 1.00 Intra-operative PRBC 76/348 (25%)) 13/49 (27%) 0.46Intra-operative FFP 5/349 (1%) 2/49 (4%) 0.21 Intra-operative platelets11/349 (3%) 3/49 (6%) 0.40 Post-operative PRBCs 150/348 (43%) 26/49(53%) 0.19 Post-operative FFP 41/349 (12%) 5/49 (10%) 0.75Post-operative platelets 47/349 (13%) 5/49 (10%) 0.53 Intra-operative2/349 (1%) 0/49 (0%) 1.00 hypotension >5 minutes Post-operative 2/349(1%) 2/49 (4%) 0.08 hypotension >5 minutes Re-sternotomy for 12/349 (3%)5/49 (10%) 0.05* bleeding Intubation time ‡ (hr) 20 (14-24) 19 (11-24)0.16 CSICU discharge in 48 hrs 315/347 (91%) 37/49 (76%) 0.001**Abbrevations used in this table: standard deviation (SD); fresh frozenplasma (FFP) packed red blood cells (PRBCs); intra-aortic balloon pump(IABP); cardiac surgical intensive care unit (CSICU); cardiopulmonarybypass (CPB); angiotensin converting enzyme inhibitor (ACEI);angiotensin-2 (AG-II). *P < 0.05 **P < 0.01; # in 362 patients on CPB;†in 359 patients on cross clamp ‡ Median (interquartile range)2. Cytokine Results

Cytokine results are illustrated in Table 2. There were significantlylower post-operative 24 hour urinary IL1ra and TNFsr2 levels in thosepatients who developed late (day 5) renal dysfunction (Table 2). Therelative value of lower than normal post-operative urinary IL1ra andTNFsr2 in predicting day 5 (late) renal dysfunction is demonstrated inthe relevant receiver operator characteristic (ROC) curves shown inFIGS. 1 and 2.

3. Discussion.

The main findings of this study are:

If patients are grouped into those with and without renal dysfunction inthe fifth post-operative day, the abnormal renal function group hadsignificantly lower 24 hour urinary IL1ra and TNFsr2 than the normalrenal function group.

TABLE 2 Table showing 24 hours post-operative urinary cytokineconcentrations (median and interquartile range)IQR) in patientscategorised according to MDRD estimated 25% eGFR percentage drop frompre-operative baseline at any time post-operatively (Any MDRD >25% drop)as well as on post-operative day 1 (MDRD D1 >25% drop), day 2 (MDRDD2 >25% drop) and day 5 (MDRD D5 >25% drop) (*P < 0.05). Any MDRD >25%drop No Yes Cytokine (pg ml⁻¹) n Median (IQR) n Median (IQR) P valueIL1ra (post-operative) 337 22891 (9526-48518) 40 18327 (7081-31354) 0.17TNFsr2 (post-operative) 342 20039 (9132-30693) 44 13955 (9990-21838)0.044 MDRD D1 >25% drop No Yes Cytokine (pg ml⁻¹) n Median (IQR) nMedian (IQR) P value IL1ra (post-operative) 370 22017 (9593-48060) 1314553 (6109-23168) 0.15 TNFsr2 (post-operative) 379 19345 (9280-30519)13 12967 (7926-22229) 0.22 MDRD D5 >25% drop No Yes Cytokine (pg ml⁻¹) nMedian (IQR) n Median (IQR) P value IL1ra (post-operative) 367 22687(9594-48006) 12 12332 (1777-22316) 0.02* TNFsr2 (post-operative) 37219688 (9204-30629) 16 11529 (8054-17261) 0.02* MDRD D2 > 25% drop No YesCytokine (pg ml⁻¹) n Median (IQR) n Median (IQR) P value IL1ra(post-operative) 349 21348 (9445-47883) 33 20306 (10883-46321) 0.79TNFsr2 (post-operative) 358 19449 (9178-30595) 33 13840 (9931-24404)0.16

The invention claimed is:
 1. A method of treating a subject at increasedrisk of renal dysfunction induced by at least one of physical trauma,hypotension, sepsis, and septic shock syndrome, wherein the methodincludes the step of: a) obtaining a urine sample from a subject afterthe at least one of physical trauma, hypotension, sepsis and septicshock syndrome; b) detecting the anti-inflammatory cytokine present inthe urine sample obtained in step a), wherein the anti-inflammatorycytokine is selected from the group consisting of IL-1ra, TNFsr1 andTNFsr2 and any combination thereof; c) detecting the anti-inflammatorycytokine in a post-event normal control; d) calculating the level ofanti-inflammatory cytokine detected in step b) relative to the level ofanti-inflammatory cytokine detected in step c); e) determining that thesubject has a greater than normal chance of developing renal dysfunctioninduced by physical trauma, hypotension, sepsis and/or septic shocksyndrome when the level of anti-inflammatory cytokine determined in stepb) is lower than the level of anti-inflammatory cytokine detected instep c); and f) applying therapeutic measures to treat or obviate thepredicted impending renal dysfunction to the subject wherein thetherapeutic measures are selected from maintaining a supra-normal bloodpressure; ensuring adequate tissue oxygen delivery; administration ofsteroids; renal replacement therapy; dialysis; or any combinationthereof.